Intracellular staining protocol

Manufactured by Thermo Fisher Scientific

The Intracellular Staining Protocol is a laboratory technique used to detect and analyze the presence and distribution of specific proteins or other molecules within the cells of a sample. The protocol involves the fixation and permeabilization of cells, followed by the addition of fluorescently labeled antibodies or other probes that bind to the target molecules of interest. This allows for the visualization and quantification of these intracellular components using flow cytometry or other imaging technologies.

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Lab products found in correlation

Celltrace violet, by Thermo Fisher Scientific (2 mentions) Fixable viability dye, by Thermo Fisher Scientific (1 mentions) Igg isotype control, by BioLegend (1 mentions) Brefeldin a solution, by Thermo Fisher Scientific (1 mentions) Ionomycin, by Merck Group (1 mentions) Tim 3, by BioLegend (1 mentions) Calcein am, by BioLegend (1 mentions) Hil 15, by R&D Systems (1 mentions) Golgistop, by BD (1 mentions) Golgiplug, by BD (1 mentions)

2 protocols using intracellular staining protocol

Isolation and Characterization of Tissue-Resident Immune Cells

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The dLNs were extracted with sterilized surgical equipment and crushed with frosted surfaces in ice‐cold PBS. The cell mixtures were then filtered through 75 mm cell strainers into 15 ml conical tubes. The cells were washed, counted, and then seeded into 48‐well plates. Single‐cell suspensions (1 million cells) were first incubated with anti‐mouse Fc blocking antibodies (BioLegend) for 30 min at 4°C and then coincubated with Fixable Viability Dye (Invitrogen) to exclude dead cells followed by co‐incubated with antibodies against CD45 (BioLegend), CD3 (BioLegend), CD8 (Invitrogen), PD‐1 (BioLegend) and Tim‐3 (BioLegend) for 20 min at 4°C. For intracellular Gzm B (Invitrogen) staining, cells were stimulated for 4 h at 37°C in a medium containing PMA (Sigma, 50 ng ml⁻¹), ionomycin (Sigma, 1 μg ml⁻¹), and brefeldin A solution (Invitrogen), and then the cells were subjected to an intracellular staining protocol (Invitrogen). Intranuclear Ki‐67 (BioLegend) staining was carried out with fixation/permeabilization buffer solution (Invitrogen) according to the manufacturer's instructions. Flow cytometry was performed with an ACEA NovoCyte, and the data were analysed with NovoExpress software. The negative staining threshold was obtained with IgG isotype control (BioLegend).

Fei X., Li Z., Yang D., Kong X., Lu X., Shen Y., Li X., Xie S., Wang J., Zhao Y., Sun Y., Zhang J., Ye Z., Wang J, & Cai Z. (2021). Neddylation of Coro1a determines the fate of multivesicular bodies and biogenesis of extracellular vesicles. Journal of Extracellular Vesicles, 10(12), e12153.

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NK Cell Activation and Cytotoxicity Assay

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NK cells were stimulated with hIL-18 (100 ng/mL, R&D), hIL-15 (20 ng/mL, R&D), and hIL-12 (10 ng/mL, BioLegend) for 18hr, washed 3 times, then cultured in cRPMI for 2 days.
NKL cells were rested in cytokine-free media for 2 days. The rested NKL cells were preincubated with 100nM surrogate ligand or hIL-2 for 12h. K562 cells were labeled with 15μM Calcein-AM (BioLegend) for 30min at 37°C. The NKL cells were cocultured with 10,000 K562 cells at indicated effector:target ratios for 4h at 37°C in V bottom 96 well plate. The supernatants were transferred to a new 96 well plate and measured using a Spectramax Gemini dual-scanning microplate (excitation filter: 485 ± 9 nm; band-pass filter: 530 ± 9 nm).
For degranulation and activation of NKL or primary NK cells, cells were rested and pre-stimulated with surrogate agonists for 12h. K562 cells were labeled with 1uM CellTrace Violet (Thermo Fisher) for 20min at 37°C. NK cells were co-cultured with K562 cells for 4h in the presence of FITC-CD107 antibody (BioLegend), GolgiStop and GolgiPlug (BD). The cells were surface stained with NK markers and CD69 antibody for 30min. on ice. IFNγ staining was performed by following the intracellular staining protocol (Invitrogen). The samples were analyzed via flow cytometry.

Yen M., Ren J., Liu Q., Glassman C.R., Sheahan T.P., Picton L.K., Moreira F.R., Rustagi A., Jude K.M., Zhao X., Blish C.A., Baric R.S., Su L.L, & Christopher Garcia K. (2022). Facile Discovery of Surrogate Cytokine Agonists. Cell, 185(8), 1414-1430.e19.

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